The present invention relates to asymmetric porous beads suitable for chromatographic use.
Biomolecules such as proteins, polypeptides and fragments of biomolecules have become important agents for pharmaceutical and diagnostic applications. Currently, the purification of biomolecules often involves multiple steps, including chromatography. The synthesis and use of porous chromatography media for biomolecule separations is well documented. Often the chromatographic steps are performed using packed beds of beads in which a product or impurity is separated from the feed stream. These beads are generally a base matrix (such as a polysaccharide, synthetic polymer or material, ceramic, glass or a composite of the foregoing) that contains or has been modified with functionality that interacts with the biomolecule via chemical or physical means; it provides a “driving force” for binding or interaction. This complex set of interactions contributes to how the media performs in the desired separation. The ability of the media to affect a given separation between biomolecules is often referred to as the “media's selectivity”. Typically these matrices exhibit porosity that allows for the biomolecule of interest to access the internal volume of the particle. This internal porosity is uniform through the particle.
Chromatographic separations typically are carried out in columns packed with the separation matrix in form of particulate beads. The size of the media particles dictates the kinetics of the separation, but smaller particles can result in high back pressure. To be able to separate large molecules the particles should have large pores, but large pores reduce the mechanical stability of the particles, particularly with polysaccharides such as agarose. Polysaccharides are advantageous because they are typically low protein binding, easy to functionalize, and can form porous structures. Conventionally, polysaccharide beads are typically made from one polysaccharide with a uniform or “symmetric” pore structure throughout the bead. Examples of these “symmetric” beads include Cellufine® (cellulose), Sepharose® (agarose) and Sephadex® (dextran). These symmetric beads bind molecules throughout the internal structure of the bead. The chemical environment (pore size, hydrophobicity, ligand type and ligand density) are essentially uniform within the internal structure. Therefore, the nature of the binding environment throughout the bead is uniform. In these systems, the driving force for separation comes from the difference in binding strength between absorbed biomolecules. Typically when a symmetric resin is used, optimization of the binding strength through control of the buffer conditions is the only driving force for achieving the desired separation. In addition, polysaccharide materials are inherently compressible, and often require chemical modification to reduce compressibility such as through crosslinking.
Another property of a symmetric resin is the mechanical strength of the media, which is a result of the material, pore structure and chemical modification. Typically for polysaccharides, the smaller the pore size of the media, the greater the mechanical strength due to higher concentration of solid material (lower porosity). However, the smaller the pore size, the more hindered the mass transfer of larger species (such as IgG) in to the adsorbent. Therefore, symmetric media design often involves the optimization/trade-off of particle rigidity and biomolecule mass transfer. This optimization problem is further complicated by the fact that the permeability of a symmetric bead is the result of the size and size distribution of the particle. As the particle size gets smaller, external particle surface area increases and therefore so does mass transfer. During elution, the smaller particle allows for a shorter diffusion time out of the media, thus causing a more concentrated, narrow elution peak. However, this improvement of the mass transfer is at the expense of permeability, which restricts column dimensions and media throughput.
In many biomolecule separations, the species of interest (target molecule) is the most concentrated species in the feedstock. In other words, the impurities are only a small percentage of the total mixture to be separated. With symmetric chromatography media, the separation is often affected by binding the target molecule and some of the impurities, and then separating the impurity by using the elution condition to differentiate the species. If the two species are very similar in their binding strength to the media, even if the proteins are different in size, the separation can be very difficult. In some cases, the difficult nature of the separation is the result of the limited number of driving forces affecting the purification. The end result is either a lower yield of purified protein from incomplete separation or that another chromatography step is then essential to further purify the target molecule using a different media with different driving forces for separation. This is time-consuming, inefficient and expensive.
Filled polysaccharide beads are well established for uses including expanded bed or fluidized bead chromatography. Typically, these materials are made by adding a solid or non-porous sphere to the polysaccharide (typically agarose) during bead formation. In this manner, a bead with one or more non-porous particles encapsulated inside the polysaccharide material can be formed. Another common technique is to coat the solid particle with a material that eventually becomes the absorbent. The solid particle serves no function in the protein capacity or separation properties of the bead. The particle typically acts only to modify the density of the bead such that the material can be used for non-packed bed applications. In some cases the solid particle provides rigidity and/or reduced gel volume.
Recently, materials have been developed with so-called “lids” or an outer layer of non-absorptive polysaccharide used to restrict the entrance to the porous structure, in order to avoid the binding of large molecules while maintaining capacity for smaller species. These materials have very low capacities for larger molecules as their internal surface area is not available to both species for binding.
Previous work has shown it possible to modify a symmetric polysaccharide structure with a chemical modification in an asymmetric way. This technique allows for the creation of a changing chemical environment within the bead. Typically the modification is used to provide a neutral layer on the outside of the bead. This prevents fouling of the outside of the bead, especially in dirty feed streams such as those found in expanded bed absorption (EBA). However, this technique does not change the pore size of the bead, therefore resulting in a symmetric pore size throughout the bead.
Methods also have been developed to modify the pore size of porous chromatography media. Using these techniques, the creation of a bimodal distribution of pore sizes is possible. However, this pore structure is evenly distributed throughout the bead and therefore does not create chemically unique regions within the bead to tune/alter observed selectivity.
Accordingly, a better media design is needed to improve biomolecule purifications in which the separation is driven by more than one characteristic of the biomolecule.